GSM6813489: N2 (WT), adult day 2, replicate 1; Caenorhabditis elegans; RNA-Seq
When animals reached day 2 of adulthood, they were washed off and collected from the plates in M9 buffer, followed by two washes in M9, and a final wash with DEPC-treated RNase-free water.  Approximately two times the volume of the animal pellet of TRIzol® reagent was added to each animal preparation, followed by brief mixing and freezing overnight at -80 °C. Tubes were then allowed to partially thaw, and were vortexed for five minutes to assist with disrupting the cuticle. Each sample was transferred to a new tube, and 200 µL of chloroform per 1 mL of TRIzol was added to each tube, followed by vortexing for 20 seconds, and the tubes were allowed to settle at room temperature for 10 minutes. The supernatants were transferred to new tubes, and an equal amount of 100% ethanol was added and mixed before proceeding to column purification with the Qiagen RNAeasy Mini kit according the manufacturer's instructions. Samples were eluted with 30 – 50 µL of DEPC-treated water, and checked for initial concentration and quality with a Nanodrop ND-1000 spectrophotometer. Isolated RNA was provided to the University of Rochester Genomics Research Center for library preparation and sequencing. Prior to library preparation, RNA integrity of all samples was confirmed on an Agilent 2100 Bioanalyzer. Libraries for sequencing were prepared with the Illumina TruSeq Stranded mRNA kit, according to the manufacturer's instructions. Quality of the resulting libraries was checked again with a Bioanalyzer prior to sequencing to ensure integrity.